Summary:ModelingEA/TEFInHumanPSC-DerivedEmbryonicTissues During development of the vertebrate embryo, a common foregut tube gives rise to the esophagus and respiratory tract and this involves an array of complex molecular and morphological processes. The dorsal foreguttubeformstheesophagusandtheventraldomainformstherespiratorytract,andfailuretodosocan resultintracheaesophagealbirthdefectssuchasesophagealatresiaandtracheoesophagealfistula(EA/TEF). As discussed in project 2, much is known about how Wnt and BMP signaling promote a respiratory fate by activation of the transcription factor Nkx2.1. In contrast, little is known about pro-esophageal factors. Mouse and human studies demonstrate that the HMG-box transcription factor Sox2 is involved in segregation of the esophageal and respiratory lineages, however whether Sox2 promotes an esophageal fate or acts predominantlytorepressrespiratory-inducingpathwaysthedorsalforegutisunclear.Wehypothesizethatboth mechanismsareinvolvedinnormalesophagealdevelopment. In humans, most genes that cause EA/TEF remain unidentified. However, heterozygous mutations in SOX2 can cause of EA and TEF, which is in contrast to mice with heterozygous loss of Sox2, which are normal. CompletelossofSox2fromtheforegutendodermofmouseembryosresultsinesophagealagenesis,however Sox2isalsoexpressedduringdevelopmentoftheentericnervoussystem(ENS)oftheesophagus.Giventhat patients with EA can have motility defects, we hypothesize some EA-associated genes may affect ENS development.However,astudyofhowEA-associatedmutationsdifferentiallyaffecttheepitheliumand/orENS of the esophagus has never been done in any species, let alone humans. We propose several novel PSC- basedapproachestostudyhowSox2andotherEA-associatedgenesimpactHumanesophagusspecification, epithelial morphogenesis, and functional innervation using human pluripotent stem cell-derived esophageal organoidswithanentericnervoussystem. In this project we aim to identify the mechanisms underlying esophageal specification and developmentinhumansbyfirstfocusingonthekeyesophagealfactorSox2.WehypothesizethatSOX2 acts both to repress the respiratory lineage, and promote an esophageal fate via an unidentified gene regulatorynetwork.WewilluseahumanPSC-derivedforegutmodelincombinationwithSOX2gain-andloss- of-function to identify a respiratory GRN that is repressed by SOX2 and an esophageal GRN that is SOX2- dependant.ConverselywewilldetermineifNKX2.1repressestheesophagealfate.Wewilltakeadvantageof theexpandablenatureofhumanforegutculturestoidentifydirecttranscriptionaltargetsofhumanSOX2and NKX2.1usingRNA-seqandChIP-seq.WewilltheninvestigatethediseasemechanismsunderlyingTEF andEAthatarecausedbySox2mutations.WewillgeneratePSClinesharboringpatient-basedmutationsin SOX2 and investigate how these impact the formation of the esophageal and respiratory lineages. We will identify the impact of SOX2 mutations on Wnt and BMP signaling and if Sox2 acts by direct protein-protein interactions with the effector proteins b-catenin/TCF and Smads. Lastly we will investigate how EA mutationsdifferentiallyeffectthedifferentcelltypesoftheesophagus;?theepithelial,smoothmuscle and ENS. Given that some patients with EA have associated motility disorders including achalasia 3, constrictions4andmegaesophagus5,wewillinvestigateifSox2mutationsalsohaveENSdeficits.Wewilluse iPSC lines derived from EA/TEF patients identified in projects 1 and 2 to model the molecular deficits underlyingthisbirthdefectusingourhumanPSC-derivedorganoidmodel.